Assay kit and method for detecting modulators of gpib-thrombin interaction

ABSTRACT

The present invention is in the field of coagulation diagnostics and relates to methods for detecting modulators of GPIb-thrombin interaction in a sample. To this end, the sample is contacted with isolated mutated GPIbα protein and thrombin, and the formation of a complex between mutated GPIbα protein and thrombin is determined.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of U.S. patent application Ser. No. 14/643,938, filedMar. 10, 2015, now U.S. Pat. No. xx,xxx,xxx, which claims priority toEuropean Patent Application No. EP 14158716.2, filed Mar. 11, 2014, thedisclosures of which are hereby incorporated by reference herein intheir entireties for all purposes, and which also incorporates byreference herein in its entirety the sequence listing which is submittedherewith in computer readable form.

FIELD

The present invention is in the field of coagulation diagnostics andrelates to assay kits and methods for detecting modulators ofGPIb-thrombin interaction in a sample.

BACKGROUND

Thrombin (factor IIa) is involved in a multiplicity of activating andinhibitory mechanisms of plasmatic blood coagulation and is thus thecentral enzyme of secondary hemostasis, which primarily comprises theformation of fibrin. Less well studied and understood is the role ofthrombin in primary hemostasis, which comprises the activation ofplatelets and adhesion of platelets as a result of an endothelialinjury. It is known that thrombin is a platelet activator and stimulatesthe aggregation of platelets. The most important thrombin receptors onthe platelet surface are, firstly, the PAR receptors (protease-activatedreceptors) and, secondly, the glycoprotein Ib-V-IX receptor complex. Theglycoprotein Ib-V-IX receptor complex comprises the integral membraneprotein glycoprotein Ib (GPIb), the integral membrane proteinglycoprotein IX (GPIX) and glycoprotein V (De Candia, E., Mechanisms ofplatelet activation by thrombin: A short history. Thrombosis Research2012, 129: 250-256).

GPIb is a double-chain molecule consisting of a heavy chain with anapparent molecular mass of about 145 kDa (synonymous: alpha-chain orGPIbα) and a light chain with an apparent molecular mass of about 22 kDa(synonymous: beta-chain or GPIbβ) which are connected to one another viadisulfide bonds (Lopez, J. A. et al., Cloning of the α chain of humanplatelet glycoprotein Ib: A transmembrane protein with homology toleucine-rich α₂-glycoprotein. Proc. Natl. Acad. Sci USA 1987, 84:5615-5619).

The GPIbα protein contains binding sites for thrombin and thus bringsabout the binding of thrombin to the glycoprotein Ib-V-IX receptorcomplex. A fragment of the GPIbα chain is glycocalicin, which isproteolytically cleaved from the intact receptor in the plateletmembrane. Glycocalicin is detectable in plasma. Elevated concentrationsof free glycocalicin in plasma indicate a disruption in plateletfunction (Beer, J. H. et al., Glycocalicin: A New Assay—The NormalPlasma Levels And Its Potential Usefulness in Selected Diseases. Blood1994, 83(3): 691-702).

Since thrombin and platelets play a central role in the development ofarterial thromboses and since inhibitors of platelet aggregation forprophylactic and therapeutic use are meanwhile being researched andused, the specific study of the interaction of thrombin and platelets isof great interest.

It is therefore desirable to have methods which allow the detection ofmodulators, i.e., inhibitors or activators, of platelet-thrombininteraction in patient samples. Such methods would allow the monitoringof platelet inhibitor therapies or even the detection of physiologicaldisruptive factors, such as, for example, activating or inhibitoryautoantibodies.

SUMMARY

It is an object of the present invention to provide a method forspecifically detecting modulators of GPIb-thrombin interaction in asample.

The object is achieved by contacting the sample with isolated GPIbαprotein and with isolated thrombin and determining the formation of acomplex between the GPIbα protein and thrombin, the GPIbα protein beingmutated and, compared to the wild-type sequence of the human GPIbαprotein, containing at least the amino acid residues 1-268 and having asubstitution Xaa at at least one of the positions 233, 235, 237 and 239(SEQ ID NO: 1).

In another aspect, an assay kit for carrying out a method for detectingmodulators of GPIb-thrombin interaction in a sample contains a firstreagent containing isolated GPIbα protein, the GPIbα protein beingmutated and, compared to the wild-type sequence of the human GPIbαprotein, contains at least the amino acid residues 1-268 and has asubstitution Xaa at at least one of the positions 233, 235, 237 and 239(SEQ ID NO: 1), and a second reagent containing thrombin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the absorbance measurement values [A] of reaction volumeswith different anti-GPIb antibodies in different concentrations. Theantibodies VM16d, SZ2 and 4H12 inhibit the thrombin-GPIb interaction ina concentration-dependent manner (see example 1).

DETAILED DESCRIPTION

The term “modulator of GPIb-thrombin interaction” encompasses substanceswhich influence the GPIb-thrombin interaction. Inhibitors ofGPIb-thrombin interaction reduce the binding of thrombin to GPIbαprotein. Activators of GPIb-thrombin interaction intensify the bindingof thrombin to GPIbα protein.

If the sample contains an activator, the formation of a complex isintensified compared to a standard sample.

If the sample contains an inhibitor, for example

-   -   a therapeutically administered thrombin inhibitor, for example        from the group of exosite I inhibitors (e.g., hirudin) or        exosite II inhibitors (e.g., heparin) (Ruggeri, Z. M. et al.,        Unravelling the mechanism and significance of thrombin binding        to platelet glycoprotein Ib. Thrombosis and Haemostasis 2010,        104.5: 894-902) or    -   a physiological thrombin inhibitor, such as, for example,        autoantibodies against thrombin, which prevent the binding of        thrombin to GPIbα, or    -   a therapeutic administered GPIbα inhibitor, such as, for        example, an anti-GPIbα antibody, for example, the antibody 4H12        (U.S. Pat. No. 5,486,361 A) or the antibody SZ2 (Ruan, C. et        al., A murine antiglycoprotein Ib complex monoclonal antibody,        SZ 2, inhibits platelet aggregation induced by both ristocetin        and collagen. Blood 1987, 69(2): 570-577) or H6B4-Fab, the Fab        fragment of a humanized monoclonal anti-GPIbα antibody        (Firbas, C. et al., Targeting von Willebrand factor and platelet        glycoprotein Ib receptor. Expert Rev. Cardiovasc. Ther. 2010,        8(12): 1689-1701), or GPG-290, a recombinant, chimeric antibody        containing the amino-terminal amino acids 1-290 of GPIbα coupled        to human IgG1 (Yeung, J. & Holinstat, M., Newer agents in        antiplatelet therapy: a review. Journal of Blood Medicine 2012,        3: 33-42), or    -   a physiological GPIbα inhibitor, such as, for example,        autoantibodies against GPIbα, which prevent the binding of GPIbα        to thrombin, or    -   elevated glycocalicin concentrations, which compete with the        GPIbα protein for binding to thrombin,

the formation of a complex is reduced compared to a standard sample.

The present invention therefore provides a method for detectingmodulators of GPIb-thrombin interaction in a sample, the sample beingcontacted with isolated GPIbα protein and with isolated thrombin and theformation of a complex between the GPIbα protein and thrombin beingdetermined. The GPIbα protein used is mutated and, compared to thewild-type sequence of the human GPIbα protein, contains at least theamino acid residues 1-268 and has a substitution Xaa at at least one ofthe positions 233, 235, 237 and 239 (SEQ ID NO: 1).

It is advantageous that said method manages without the use ofplatelets. The preparation of platelet reagents from animal or humanblood is costly and inconvenient and does not guarantee a consistentquality.

The term “sample” encompasses biological liquids particularly fromhumans and animals, such as blood, plasma or serum.

The term “standard sample” encompasses a reference material which, whenused as a sample in the method according to the invention, generates ameasurement value corresponding to the GPIb-thrombin interaction of ahealthy individual or of a healthy population of individuals, whichindividual or which population does not have a GPIb-thrombin interactioninfluenced by a modulator of GPIb-thrombin interaction. A suitablereference material is, for example, a pool composed of a body fluid, forexample, a standard plasma pool or standard serum pool, from generallyat least 20 clearly healthy individuals.

The GPIbα protein used in the method according to the invention can be arecombinantly or synthetically produced GPIbα protein. Suitable for theproduction of recombinant GPIbα protein are known prokaryotic oreukaryotic expression systems, such as, for example, the expression inbacteria (e.g., E. coli), in yeasts (e.g., Saccharomyces cerevisiae,Pichia pastoris), in plant, animal or human cell cultures. Suitable forthe production of synthetic GPIbα protein are known techniques for invitro protein synthesis, such as, for example, solid-phase syntheses(e.g., Merrifield synthesis). Preferably, the GPIbα protein used in themethod according to the invention is recombinantly produced GPIbαprotein which was produced in a culture of human cells, preferably in aculture of human embryonic kidney cells (HEK cells).

Preferably, the GPIbα protein is added to the assay volume in such anamount that a final concentration of 0.5-50 μg/mL GPIbα in the assayvolume, particularly preferably of 1-10 μg/mL GPIbα in the assay volume,very particularly preferably of 5 μg/mL GPIbα in the assay volume, isobtained.

The GPIbα protein used in the method according to the invention can, atthe N-terminus, be fused to the homologous human GPIbα signal sequenceMPLLLLLLLLPSPLHP (SEQ ID NO: 2, also referred to as amino acid residues−16 to −1). Alternatively, the GPIbα protein used can, at theN-terminus, be fused to a heterologous signal sequence, i.e., to apolypeptide not usually present in the human GPIbα polypeptide, whichsignal sequence, however, positively influences the expression and/orsecretion of the recombinantly expressed GPIbα protein in the selectedexpression system. A suitable heterologous signal sequence is, forexample,

(SEQ ID NO: 3) MPLQLLLLLILLGPGNSLQLWDTWADEAEKALGPLLARDRR.

Furthermore, the GPIbα protein used in the method according to theinvention can, at the C-terminus, be fused to one or more affinity tagswhich allow the binding of the, for example, recombinantly expressedprotein to an affinity support, allowing, for example, the purificationof recombinantly expressed GPIbα protein. Preference is given to smallaffinity tags having a length of not more than 12 amino acids.Particular preference is given to affinity tags from the groupconsisting of His-tag, Flag-tag, Arg-tag, c-Myc-tag and Strep-tag.Suitable affinity supports which bind with high affinity to an affinitytag are, for example, specific antibodies, immobilized cations (e.g.,Ni²⁺ with affinity for His-tags) or other types of binding partners(e.g., streptavidin with affinity for Strep-tags).

The isolated GPIbα protein used is mutated and—compared to the wild-typesequence of the human GPIbα protein (SEQ ID NO: 1)—contains at least theamino acid residues 1-268 and a substitution Xaa at at least one of thepositions 233, 235, 237 and 239. Preferably, the mutated GPIbα proteincontains a substitution Xaa in each case at two of the positions 233,235, 237 and 239. It was found that, surprisingly, the use of wild-typeGPIbα protein is not suitable for the detection of modulators ofGPIb-thrombin interaction.

Preferably, the substitutions Xaa of the glycine residue at position 233and of the methionine residue at position 239 of the GPIbα chain consistof a valine residue (G233V and M239V) or a serine residue (G233S andM239S). Any desired combination of different substitutions Xaa at thetwo positions is possible. Particular preference is given to thecombination G233V/M239V. The substitution Xaa of the aspartic acidresidue at position 235 preferably consists of a tyrosine residue(D235Y). The substitution Xaa of the lysine residue at position 237preferably consists of a valine residue (K237V). The stated mutationsare gain-of-function mutations which are known to have a significantlyhigher affinity for VWF and interact more strongly with VWF thanwild-type GPIbα protein. Neither ristocetin, botrocetin nor aristocetin-equivalent substance is added to the assay volume.

The thrombin used in the method according to the invention can berecombinant human or bovine thrombin, or human or bovine thrombinisolated from natural sources.

In a preferred embodiment of the method according to the invention, thethrombin and/or the GPIbα protein is associated with a solid phase.

The term “associated” can be broadly comprehended and encompasses, forexample, a covalent and a noncovalent bond, a direct and an indirectbond, the adsorption to a surface and the containment in a recess. Inthe case of a covalent bond, the isolated GPIbα protein is bonded to thesolid phase via a chemical bond. An example of a noncovalent bond issurface adsorption. Besides a direct bond to the solid phase, theisolated GPIbα protein or the thrombin can also be indirectly bonded tothe solid phase via a specific interaction with other specific bindingpartners, for example, via a specific interaction with an antibody or anantibody fragment, preferably with an anti-GPIbα antibody or with ananti-thrombin antibody or—if the isolated protein has an affinitytag—with an anti-affinity-tag antibody.

In the context of this invention, the term “solid phase” includes anarticle which consists of porous and/or nonporous, water-insolublematerial and can have a very wide variety of different forms such as,for example, vessel, tube, microtitration plate (ELISA plate), bead,microparticle, rod, strip, filter or chromatography paper, etc.Generally, the surface of the solid phase is hydrophilic or can be madehydrophilic. The solid phase can consist of a very wide variety ofdifferent materials such as, for example, of organic and/or of inorganicmaterials, of synthetic materials, of naturally occurring materialsand/or of modified naturally occurring materials. Examples ofsolid-phase materials are polymers such as, for example, cellulose,nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide,crosslinked dextran molecules, agarose, polystyrene, polyethylene,polypropylene, polymethacrylate or nylon; latex; ceramics; glass;metals, in particular noble metals such as gold and silver; magnetite;mixtures or combinations of the same. The term “solid phase” explicitlydoes not encompass cells, in particular platelets (thromobocytes). Thus,in any case, the solid phase is a nonthrombocytic solid phase.

The solid phase can have a coating composed of one or more layers, forexample, composed of proteins, carbohydrates, lipophilic substances,biopolymers, organic polymers or mixtures thereof, in order, forexample, to suppress or prevent the nonspecific binding of sampleconstituents to the solid phase or in order, for example, to achieveimprovements with respect to the suspension stability of particulatesolid phases, with respect to storage stability, with respect toshape-giving stability or with respect to resistance against UV light,microbes or other destructively acting agents.

Contacting of isolated GPIbα protein with thrombin leads to theformation of a complex composed of the two components. If the patientsample which is added contains substances which influence said complexformation, for example, GPIb or thrombin inhibitors or GPIb or thrombinactivators, a complex formation which is altered with respect to thestandard is measured. The standard is determined by determining theGPIb-thrombin interaction in suitable reference materials, for example,in a standard plasma pool, and can, for example, be defined as 100% ofthe norm. The GPIb-thrombin interaction which is determined in a samplefrom an individual can then be set in relation to the reference value.

In one embodiment of the method according to the invention, at least oneof the two components, i.e., GPIbα and/or thrombin, is associated with aparticulate solid phase, preferably with latex particles. The formationof a complex between thrombin, GPIbα protein and the associated solidphase(s) can then be determined by measuring the agglutination of theparticulate solid phase. To quantitatively determine the agglutinationreaction, which correlates with the formation of a complex, use can bemade of, for example, light scattering on the particle aggregates viathe measurement of scattered light intensity (nephelometry) or via themeasurement of turbidity of the medium (turbidimetry).

In another embodiment of the method according to the invention, each ofthe two components, i.e., GPIbα and thrombin, is or becomes associatedwith a first and a second component of a signal-forming system, whichcooperate in such a way that a detectable signal is produced when thefirst and the second component of the signal-forming system are broughtinto close proximity with one another. A cooperation between thecomponents is to be understood in particular to mean an energy transfer,i.e., the direct transfer of energy between the components, for example,by means of light radiation or electron radiation and also via reactivechemical molecules, such as, for example, short-lived singlet oxygen.The energy transfer can take place from one component to anothercomponent; however, another possibility is a cascade of differentsubstances, via which the energy transfer proceeds. For example, thecomponents can be a pair composed of an energy donor and an energyrecipient, such as, for example, photosensitizer and chemiluminescentagent (EP-A2-0515194, LOCI® technology) or photosensitizer andfluorophore (WO-A1-95/06877) or radioactive iodine<125> and fluorophore,or fluorophore and fluorescence quencher.

In another embodiment of the method according to the invention, thethrombin is associated with a nonparticulate solid phase, preferablywith the surface of a microtiter plate. The formation of a complexbetween thrombin and GPIbα protein can then be determined by measuringthe amount of GPIbα which is bound to the solid phase via the thrombin.To determine the amount of GPIbα which has been bound to the solid phasevia the thrombin, it is, for example, possible to use an anti-GPIbαantibody which is directly or indirectly associated with a component ofa signal-forming system and thus allows the quantification of the amountof GPIbα bound. Alternatively, the GPIbα protein can be associated witha nonparticulate solid phase, and the formation of a complex betweenthrombin and GPIbα protein can be determined by measuring the amount ofthrombin which is bound to the solid phase via the GPIbα protein. Todetermine the amount of thrombin which has been bound to the solid phasevia the GPIbα, it is, for example, possible to use an anti-thrombinantibody which is directly or indirectly associated with a component ofa signal-forming system or a peptide substrate having athrombin-cleavable signal group, for example, a chromogenic, fluorogenicor electrogenic signal group.

The present invention further provides an assay kit for carrying out amethod according to the invention, containing a first reagent containingisolated GPIbα protein, the GPIbα protein being mutated and, compared tothe wild-type sequence of the human GPIbα protein, containing at leastthe amino acid residues 1-268 and having a substitution Xaa at at leastone of the positions 233, 235, 237 and 239 (SEQ ID NO: 1), and a secondreagent containing thrombin. Particular preference is given to an assaykit containing a reagent containing isolated, mutated GPIbα proteinwhich has a substitution Xaa in each case at at least two of thepositions 233, 235, 237 and 239, particularly preferably at thepositions 233 and 239. Very particularly preferably, the substitutionsXaa of the glycine residue at position 233 and of the methionine residueat position 239 of the GPIbα chain consist of a valine residue (G233Vand M239V). Another preferred assay kit contains a reagent containingisolated, mutated GPIbαprotein which has a substitution Xaa in each caseat the positions 233, 235 and 239. Preferably, the substitutions Xaa ofthe glycine residue at position 233 and of the methionine residue atposition 239 of the GPIbα chain consist of a valine residue (G233V andM239V) or a serine residue (G233S and M239S) and the substitution Xaa ofthe aspartic acid residue at position 235 consists of a tyrosine residue(D235Y).

In one embodiment of the assay kit, the second reagent can comprise asolid phase to which the thrombin is associated. Preferably, such anassay kit further contains one further reagent or two or more furtherreagents for detecting the isolated GPIbα protein, containing, forexample, an anti-GPIbα antibody or an anti-Tag antibody which isdirectly or indirectly labeled with an enzyme and a substrate for theenzyme, for example, a horseradish peroxidase-labeled antibody and thechromogenic substrate tetramethylbenzidine.

In another embodiment of the assay kit, the first reagent can comprise asolid phase to which the mutated GPIbα protein is associated.Preferably, such an assay kit further contains one further reagent ortwo or more further reagents for detecting thrombin, containing, forexample, an anti-thrombin antibody which is directly or indirectlylabeled with an enzyme and a substrate for the enzyme, for example, ahorseradish peroxidase-labeled antibody and the chromogenic substratetetramethylbenzidine. Alternatively, it is also possible to use apeptide substrate having a thrombin-cleavable signal group in order todetect thrombin.

The reagents can be provided in liquid or lyophilized form. If a reagentis present as a lyophilisate, the assay kit can additionally contain asolvent required for suspending the lyophilisate, such as, for example,distilled water or a suitable buffer.

EXAMPLES Example 1 Detection of Inhibitory GPIb Antibodies in a Sample

A microtiter plate was coated with antibodies against human thrombin.Human thrombin was obtained from Sigma-Aldrich (T7009, Sigma-Aldrich,Hamburg, Germany). 100 μL of a solution of 1 μg/mL thrombin in glycerolbuffer (8 mL of distilled water, 87.7 mg of NaCl, 69 mg of NaH₂PO₄*H₂O,813 μL of 87% strength glycerol, 18 μL of Tween® 20, 10 mg of bovinealbumin, 1 mg of bovine IgG, 2 mg of phenol, 18.6 mg of Titriplex I,adjusted to pH 6.8 with ca. 43 μL of 10 N NaOH) were added to each welland incubated for one hour at room temperature. This was followed bywashing four times with 300 μL of wash buffer.

Use was made of a recombinantly produced, Flag-tag-fused GPIbα protein(aa 1-268) in which the glycine residue at position 233 and themethionine residue at position 239 is replaced in each case by a valineresidue (G233V, M239V). From this GPIbα protein, 200 μL of a 10 μg/mLsolution in glycerol buffer were mixed in each case with 200 μL of theinhibitory anti-GPIbα antibodies 4H12, SZ2 and VM16D or of the controlantibody AK2 in different concentrations in glycerol buffer andincubated for one hour and 10 minutes at room temperature. From theseGPIbα protein/antibody mixtures, 100 μL were pipetted in each case intoa well of the microtiter plate and incubated for one hour at roomtemperature. This was followed by washing four times with 300 μL of washbuffer.

To quantitatively detect the bound, Flag-tag-fused GPIbα protein, 100 μLof a 0.06 μg/mL solution of anti-Flag M2-peroxidase (Sigma-Aldrich,Hamburg, Germany) in glycerol buffer were added in each case to eachwell and incubated for one hour at room temperature. After washing fourtimes with 300 μL of wash buffer, 100 μL of a solution of thechromogenic peroxidase substrate TMB (tetramethylbenzidinedihydrochloride) and hydrogen peroxide were added to each well andincubated for 20 minutes. The reaction was stopped, and the absorbanceof the reaction volumes was measured with light at a wavelength of 450nm in an ELISA plate reader using a reference wavelength of 650 nm.

The absorbance measurement values are shown in FIG. 1.

The anti-GPIb antibody AK2 was used as control antibody, which is knownnot to influence the binding of thrombin to the GPIbα protein, but torather inhibit the ristocetin-induced binding of VWF to the GPIbαprotein (Ward, C. M. et al., Mocarhagin, a novel cobra venommetalloproteinase, cleaves the platelet von Willebrand factor receptorglycoprotein Ibα. Identification of the sulfated tyrosine/anionicsequence Tyr-276-Glu-282 of glycoprotein Ibα as a binding site for vonWillebrand factor and α-thrombin. Biochemistry 1996, 35: 4929-4938).

It is known that the anti-GPIb antibody 4H12 inhibits the binding ofthrombin to the GPIbα protein very strongly (Gralnick, U.S. Pat. No.5,486,361 A).

The anti-GPIb antibody SZ2 (Ruan, C. et al., 1987) is in development asan inhibitory anti-platelet therapeutic (Yeung, J. & Holinstat, M.,2012).

The anti-GPIb antibody VM16d is likewise known as an antibody whichinhibits the binding of thrombin to GPIb (Dubois, C. et al., Thrombinbinding to GPIbα induces integrin αIibβ3 dependent platelet adhesion tofibrin in ex vivo flowing whole blood. Thromb Haemost 2004, 91:233-237).

As is apparent from FIG. 1, 4H12, SZ2 and VM16d inhibit theGPIbα-thrombin interaction in a concentration-dependent manner in themethod according to the invention. The method is therefore suitable fordetecting inhibitors of GPIb-thrombin interaction in a sample.

What is claimed is:
 1. An assay kit for carrying out a method fordetecting modulators of glycoprotein Ib (GPIb)-thrombin interaction in asample, the assay kit containing a first reagent containing isolatedGPIbα protein, the GPIbα protein being mutated and, compared to thewild-type sequence of the human GPIbα protein, containing at least theamino acid residues 1-268 and having a substitution Xaa at at least oneof the positions 233, 235, 237 and 239 (SEQ ID NO: 1), and a secondreagent containing thrombin.
 2. The assay kit of claim 1, wherein thesecond reagent comprises a solid phase to which the thrombin isassociated.
 3. The assay kit of claim 1, further containing one or morereagents for detecting the isolated GPIbα protein.
 4. The assay kit ofclaim 3, wherein the reagent(s) for detecting the isolated GPIbα proteincomprise(s) an enzyme-labeled antibody specific for the isolated GPIbαprotein and a substrate for the enzyme.
 5. The assay kit of claim 1,wherein the first reagent comprises a solid phase to which the isolatedGPIbα protein is associated.
 6. The assay kit of claim 1, furthercontaining one or more reagents for detecting thrombin.
 7. The assay kitof claim 6, wherein the reagent(s) for detecting thrombin comprise(s)either an enzyme-labeled antibody specific for thrombin and a substratefor the enzyme or a peptide substrate having a thrombin-cleavable signalgroup.